Antibody Response to Symptomatic Infection With SARS‐CoV‐2 Omicron Variant Viruses, December 2021–June 2022

ABSTRACT We describe humoral immune responses in 105 ambulatory patients with laboratory‐confirmed SARS‐CoV‐2 Omicron variant infection. In dried blood spot (DBS) collected within 5 days of illness onset and during convalescence, we measured binding antibody (bAb) against ancestral spike protein receptor binding domain (RBD) and nucleocapsid (N) protein using a commercial multiplex bead assay. Geometric mean bAb concentrations against RBD increased by a factor of 2.5 from 1258 to 3189 units/mL and by a factor of 47 against N protein from 5.5 to 259 units/mL between acute illness and convalescence; lower concentrations were associated with greater geometric mean ratios. Paired DBS specimens may be used to evaluate humoral response to SARS‐CoV‐2 infection.


| Introduction
Humoral immune responses to infection with SARS-CoV-2 include production of immunoglobulin G (IgG) antibodies that bind to spike (S) glycoprotein, including the receptor binding domain (RBD) within S, as well as the nucleocapsid (N) protein.
Virus neutralization titers and S protein binding antibodies have been associated with protection against symptomatic infection with ancestral and pre-Omicron SARS-CoV-2 variants [1][2][3][4].Because antibodies to N protein are not elicited by US-licensed COVID-19 vaccines, the presence of anti-N antibodies can be an indicator of past SARS-CoV-2 infection among vaccinated and unvaccinated individuals [5,6].Elevated levels of anti-N antibody may indicate more recent SARS-CoV-2 infection [7].As new SARS-CoV-2 variants have emerged, serologic assays that quantify IgG antibody binding to S and N protein antigens have been used to evaluate humoral response to SARS-CoV-2 infection [8].Postinfection antibodies may reduce risk of reinfection with new SARS-CoV-2 variants, suppress viral replication, and reduce COVID-19 disease severity following reinfection [2,8].
DBS specimens have been validated as alternatives to venous blood for use with multiple SARS-CoV-2 binding antibody assays [9][10][11].Using DBS specimens collected during acute illness from patients with laboratory-confirmed COVID-19 and non-COVID-19 illnesses, we previously showed that anti-N antibody seropositivity modified COVID-19 mRNA vaccine effectiveness against symptomatic SARS-CoV-2 infection with SARS-CoV-2 Delta and Omicron variants [12].Among acutely ill patients, higher levels of binding antibodies against ancestral S protein reduced the odds of testing positive for SARS-CoV-2 Delta and Omicron variants [13].
Here, we assessed humoral immune response to SARS-CoV-2 infection comparing acute-and convalescent-phase IgG antibody levels against N and ancestral S RBD antigens among case patients infected during Omicron variant-predominant periods from December 2021 through June 2022.
Respiratory swabs and acute-phase DBS were obtained < 5 days after symptom onset from ambulatory patients with respiratory illness enrolled in the US Influenza Vaccine Effectiveness Network [12,13].Previous analysis demonstrated no increase in mean anti-N antibody concentration from DBS collected < 5 days after symptom onset among SARS-CoV-2-infected patients [13].Patients who tested positive for SARS-CoV-2 by nucleic acid amplification in respiratory specimens were scheduled for convalescent-phase blood sample collection at 21-56 days after enrollment.Data collected from enrolled participants included patient age, date of illness onset, symptoms associated with COVID-19-like illness, self-reported presence of specified underlying medical conditions, documented COVID-19 vaccination dates, self-reported laboratoryconfirmed COVID-19 < 90 or ≥ 90 days before current illness and electronic medical record of positive COVID-19 tests.
Methods for estimating SARS-CoV-2 bAb concentration from DBS have been previously published [11].DBS specimens were tested for IgG antibodies against SARS-CoV-2 recombinant antigens representing ancestral S protein RBD and N protein using a validated multiplex bead assay (FlexImmArray™ SARS-CoV-2 Human IgG Antibody Test, Tetracore, Rockville, MD) on a Luminex MAGPIX instrument with LX200 flow analyzer (Luminex Corporation, Austin, TX) [14].Eluted specimens were diluted 1:300, and individual specimen median fluorescence intensity (MFI) ratios were calculated compared to antigen-specific human IgG calibrator serum.MFI units were standardized to binding antibody unit per milliliter (BAU/mL) against World Health Organization (WHO) international standards [9,13].
Geometric mean bAb concentration (GMC), geometric mean ratios of bAb concentrations at follow-up versus enrollment, and 95% confidence interval (CI) were calculated on the log 10 scale and back-transformed to original units.We interpret geometric mean ratios as the magnitude of an immune response to ancestral S RBD and N protein following natural infection.Associations between mean bAb ratios and patient characteristics, vaccination status, baseline N antibody serostatus, and prior positive COVID-19 test were assessed by t-test or ANOVA.Statistical analysis was performed using R version 4.0.3(R Foundation for Statistical Computing, Vienna, Austria).
Increased convalescent anti-N bAb levels among baseline seropositive patients suggested boosting of immune responses acquired from past infection; this boosting was also observed among patients with ≥ 1 documented prior COVID-19 positive test.In contrast, lower baseline anti-RBD antibody concentrations among patients who had received 2 versus ≥ 3 mRNA vaccine doses were associated with greater increase in antibody response but similar convalescent antibody concentrations.While anti-RBD bAb levels correlate with protection from infection with pre-Omicron variants [1,3,4], antibody levels measured against Omicron variants were greatly reduced [8,15].Collection of DBS for SARS-CoV-2 serology was performed as part of a COVID-19 vaccine effectiveness study using a testnegative design.Finger stick blood collection was widely acceptable and provided acute-phase specimens from pediatric and adult outpatients without requiring venipuncture.We used a Tetracore multiplex bead assay that was validated by the manufacturer with sera and standardized in our laboratory for bAb quantification from DBS [13,14].While the use of plasma in serologic assays has shown a lower limit of antibody detection [10], paired DBS specimens collected during acute illness and 3-8 weeks after illness may be adequate for assessing antibody responses following natural infection.In this study, paired DBS specimens were tested using the same multiplex bead assay to estimate absolute increase in bAb concentrations.Paired specimens also facilitate comparison between humoral responses in individual patients, including those with vaccine breakthrough infections.Vaccination status and preexisting antibody levels may affect antibody response by altering viral load in early illness [8,16].Measurement of immune response to infection and antibody levels after convalescence could improve understanding of vaccine breakthrough cases and hybrid immunity [17].
The study had several limitations.The serologic assay used in our study contained ancestral SARS-CoV-2 antigens, and concentrations were converted to binding antibody units using WHO international serum standards from early in the COVID-19 response.Against pre-Omicron variants, virus neutralization titers and IgG antibody concentrations were associated with protection; however, antibody binding concentrations and neutralization activity were lower against Omicron variants [2,8,18].In addition, anti-N bAb seropositivity cut-off values were based on mean fluorescence intensity using serum standards rather than DBS [9,13].All case patients included in this study had mild illness; baseline antibody levels and immune responses may differ among patients with severe or prolonged SARS-CoV-2 infection [19].Among patients with past infection, initial anti-N bAb concentrations may have waned below the seropositivity thresholds [20][21][22].Finally, this analysis included only four unvaccinated case patients, limiting ability to compare unvaccinated infections and reinfections with vaccine breakthrough infections.
Serologic assays that quantify anti-SARS-CoV-2-specific antibody levels during and following acute infection may provide information on incidence and recency of infection.As new SARS-CoV-2 variants emerge, frequent updates to serologic antigens will be needed to quantify binding IgG antibody levels that correlate with immune protection [15].Observational studies will be critical for evaluating immune responses to COVID-19 vaccination and infection.

TABLE 1 |
Concentrations and mean increase in binding antibodies against SARS-CoV-2 nucleocapsid and ancestral spike receptor binding domain antigens during acute-and convalescent-phase of symptomatic infection during Omicron-predominant variant period.

-CoV-2 spike receptor binding domain antibody α-SARS-CoV-2 nucleocapsid antibody Geometric mean concentration, BAU/mL (95% CI)
of a serious chronic medical condition such as heart disease, lung disease, diabetes, cancer, liver or kidney disease, immune suppression, or high blood pressure.Missing n positive test indicates electronic medical record documentation of prior positive RT-PCR SARS-CoV-2 test results; no prior test indicates that no positive RT-PCR SARS-CoV-2 test was documented in electronic health records.
Abbreviation: BAU, binding antibody unit.a Student t-test or ANOVA test of difference between log transformed geometric mean ratio; p < 0.05 defined as statistically significant difference between groups.b Self-reported presence d Prior COVID-19